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Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

No MeSH data available.


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Rate of Cd2+ inhibition (on rate) on the I225W/V490W mutant of CLC-0. (A) Original recording traces from one patch using the two-pulse (to −100 mV) protocol described in Materials and methods. [Cd2+] = 100 µM. Vhold = −40 mV. (B) Combining the recording traces from A shows the time course of Cd2+ inhibition. Dotted curve is the single-exponential fit of the current amplitude elicited at various time spots except the one at 0.2 s (the second pulse). Notice the delay of Cd2+ inhibition shown by comparing the second pulse (in red) with the entire inhibition time course. (C) Delay of current inhibition upon Cd2+ application obtained at different voltages. [Cd2+] = 300 µM. Solid curve is a voltage-dependent exponential fit for the relation between the delay time and the voltage, with an estimated electric distance of −0.025. n = 7. (D) [Cd2+] dependence of the delay of current inhibition. Vm = −80 mV. Data points are the average of five to eight experiments (mean ± SEM).
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fig12: Rate of Cd2+ inhibition (on rate) on the I225W/V490W mutant of CLC-0. (A) Original recording traces from one patch using the two-pulse (to −100 mV) protocol described in Materials and methods. [Cd2+] = 100 µM. Vhold = −40 mV. (B) Combining the recording traces from A shows the time course of Cd2+ inhibition. Dotted curve is the single-exponential fit of the current amplitude elicited at various time spots except the one at 0.2 s (the second pulse). Notice the delay of Cd2+ inhibition shown by comparing the second pulse (in red) with the entire inhibition time course. (C) Delay of current inhibition upon Cd2+ application obtained at different voltages. [Cd2+] = 300 µM. Solid curve is a voltage-dependent exponential fit for the relation between the delay time and the voltage, with an estimated electric distance of −0.025. n = 7. (D) [Cd2+] dependence of the delay of current inhibition. Vm = −80 mV. Data points are the average of five to eight experiments (mean ± SEM).

Mentions: Examining the kinetics of Cd2+ inhibition on the I225W/V490W mutant is challenging because monitoring the channel current requires negative voltage pulses that relieve the binding of the Cd2+. We therefore designed a two-pulse protocol for studying the kinetics of Cd2+ inhibition. To evaluate the inhibition rate of intracellular Cd2+, a −100-mV voltage pulse for 50 ms was first given to obtain the initial current in the absence of Cd2+, followed by exposing the patch to various concentrations of Cd2+ at 0, −40, or −80 mV for various periods of time before the same pulse was given again to measure the remaining current (Fig. 12 A). Combining multiple traces from experiments using this two-pulse protocol generates the time course of the Cd2+ inhibition (Fig. 12 B). To measure the recovery rate of Cd2+ inhibition, a 50-ms voltage pulse to −100 mV was given to obtain the control current, followed by exposing the patch to 300 µM Cd2+ for 2 s. Cd2+ was then washed out for various amounts of time at 0, −40, or −80 mV before a second voltage pulse was given to assess the current. The current recorded from the second pulse was normalized to the control current before Cd2+ inhibition.


Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Rate of Cd2+ inhibition (on rate) on the I225W/V490W mutant of CLC-0. (A) Original recording traces from one patch using the two-pulse (to −100 mV) protocol described in Materials and methods. [Cd2+] = 100 µM. Vhold = −40 mV. (B) Combining the recording traces from A shows the time course of Cd2+ inhibition. Dotted curve is the single-exponential fit of the current amplitude elicited at various time spots except the one at 0.2 s (the second pulse). Notice the delay of Cd2+ inhibition shown by comparing the second pulse (in red) with the entire inhibition time course. (C) Delay of current inhibition upon Cd2+ application obtained at different voltages. [Cd2+] = 300 µM. Solid curve is a voltage-dependent exponential fit for the relation between the delay time and the voltage, with an estimated electric distance of −0.025. n = 7. (D) [Cd2+] dependence of the delay of current inhibition. Vm = −80 mV. Data points are the average of five to eight experiments (mean ± SEM).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664824&req=5

fig12: Rate of Cd2+ inhibition (on rate) on the I225W/V490W mutant of CLC-0. (A) Original recording traces from one patch using the two-pulse (to −100 mV) protocol described in Materials and methods. [Cd2+] = 100 µM. Vhold = −40 mV. (B) Combining the recording traces from A shows the time course of Cd2+ inhibition. Dotted curve is the single-exponential fit of the current amplitude elicited at various time spots except the one at 0.2 s (the second pulse). Notice the delay of Cd2+ inhibition shown by comparing the second pulse (in red) with the entire inhibition time course. (C) Delay of current inhibition upon Cd2+ application obtained at different voltages. [Cd2+] = 300 µM. Solid curve is a voltage-dependent exponential fit for the relation between the delay time and the voltage, with an estimated electric distance of −0.025. n = 7. (D) [Cd2+] dependence of the delay of current inhibition. Vm = −80 mV. Data points are the average of five to eight experiments (mean ± SEM).
Mentions: Examining the kinetics of Cd2+ inhibition on the I225W/V490W mutant is challenging because monitoring the channel current requires negative voltage pulses that relieve the binding of the Cd2+. We therefore designed a two-pulse protocol for studying the kinetics of Cd2+ inhibition. To evaluate the inhibition rate of intracellular Cd2+, a −100-mV voltage pulse for 50 ms was first given to obtain the initial current in the absence of Cd2+, followed by exposing the patch to various concentrations of Cd2+ at 0, −40, or −80 mV for various periods of time before the same pulse was given again to measure the remaining current (Fig. 12 A). Combining multiple traces from experiments using this two-pulse protocol generates the time course of the Cd2+ inhibition (Fig. 12 B). To measure the recovery rate of Cd2+ inhibition, a 50-ms voltage pulse to −100 mV was given to obtain the control current, followed by exposing the patch to 300 µM Cd2+ for 2 s. Cd2+ was then washed out for various amounts of time at 0, −40, or −80 mV before a second voltage pulse was given to assess the current. The current recorded from the second pulse was normalized to the control current before Cd2+ inhibition.

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

No MeSH data available.


Related in: MedlinePlus